The rubella virus (RV) is a togavirus and the sole representative of the Rubivirus subgroup. The small enveloped virus has a size of 65 nm and consists of a 10 kb single-stranded RNA molecule encapsulated in an icosahedral nucleocapsid which is surrounded by a lipid envelope. The rubella virus causes a relatively mild childhood disease (German measles) which usually results in permanent immunity thought to be mediated by both T-lymphocytes and antibodies. Possible consequences of rubella infections in adults are transient/chronic arthritis, musculoskeletal syndromes, insulin-dependent diabetes mellitus and late onset of neurologic sequelae.
The rubella virus is a main parameter during early pregnancy. The specific detection of anti-rubella IgM and/or anti-rubella IgG, respectively, is crucial for the clarification if an acute infection or a blazing reinfection occurred.
Maternal rubella virus infection during pregnancy is associated with a risk of congenital rubella syndrome (CRS) in the fetus, the incidence of congenital malformations being highest when primary infection occurs in the first 12 weeks of gestation. For this reason the prevention of congenital abnormalities caused by RV infection during early pregnancy requires the determination of an individual's immune status by serology, which means a selective determination of IgG and IgM antibodies specific for rubella virus antigens. Primary rubella infection is associated with a specific IgM antibody response, while elevated levels of IgG in the absence of detectable IgM indicate an immune status which is protective against acute rubella virus infection.
When prenatal screening indicates that a woman has acquired a primary rubella infection during early stage of pregnancy, a therapeutic abortion is often recommended. As a result, it is imperative that the test results are accurate.
For detecting antibodies to RV the routine laboratory diagnosis is mainly based on ELISA (enzyme-liked immunoabsorbent assay) tests, while the most widely accepted method for determination of RV immune status in central Europe is the hemagglutination inhibition (HAI) test for the verification of the RV-IgG ELISA (Weber et al., Bull Soc Sci Med Grand Duche Luxemb. (1997), 31-41).
The detection of specific antibodies of a certain immunglobulin class can be performed by binding the immunoglobulin to a solid phase to which to specific antigen has been immobilized. The bound immunoglobulin is subsequently detected by a labeled antibody specific for human imunoglobulins of a certain class. This assay format can only be carried out by a two step assay format allowing a washing step which eliminated unbound immunoglobulins prior to detection. A one-step assay format often realized in automatic immunoassay analyzers requires the direct assay format of a double antigen sandwich, i.e. the specific antibody forms an immunocomplex binding to a first antigen which is immobilized to a solid phase or will mediate immobilization to a solid phase and to a second antigen carrying a label thus allowing quantitative or qualitative detection of the specifically bound antibody.
The selective determination of specific IgG antibodies in the presence of IgM antibodies of the same specificity in a one-step double antigen sandwich format strictly requires the use of soluble, monomeric or defined oligomeric antigens (EP 944,838), which reveals an immunoreactive conformation.
The rubella virus harbors four structural proteins which have been shown to be antigenic in animals and humans. These are the three envelope glycoproteins E1 (58 kDa), E2a (47 kDa) and E2b (42 kDa) and the non-glycosylated capsid (C) protein (33kDa) decorating the single RNA plus strand which constitutes the viral genome (Waxham and Wolinsky, Rev Infect Dis. (1985) 133-9; Oker-Blom et al., J Virol. 1984 (2):403-8).
It was shown that E2a and E2b are variants of the same gene product and the difference in migration in polyacrylamide gels is due to heterogenous glycosylation of the proteins. E1 and E2 have been found to form monomers or disulfide-linked complexes (E1-E1 and E1-E2), whereas C exists exclusively as a homodimer (Waxham and Wolinsky, Virology. (1983) 126 (1), 194-203; Wolinsky et al., Rubella (1996) Fields Virology. Lippincott-Raven Publisher, Philadelphia, 899-929). An extensive review of the biological, physical and biochemical properties of RV as well as the clinical features of the infection has been published by Wolinsky (1996).
At present, antigens are used for the detection of a rubella infection, which are derived from stably infected/transfected cell-lines and, in general, stem from eucaryotic overproduction. Seppanen et al., J. Clin. Microbiol (1991) 1877-1882 describes the expression of E1 and E2 of the rubella virus in Spodoptera frugiperda Sf9 insect cells by using the baculovirus expression system. Furthermore, a stably transfected CHO cell line expressing and secreting the structural proteins E1, E2 and C of RV in the form of RV-like particles (RLPs) is disclosed in Hobman et al., 1994 (574-585) Virology.
Rubella-like particles are composed of the rubella main-antigens E1, E2 and C, which are expressed as a viral polypeptide precursor protein. Due to signal sequences this unprocessed precursor protein is secreted into the media and virus-like particles are formed. The surface of these RLPs presents epitopes suited for the immunological detection of anti-RV antibodies in rubella-positive sera. The expression of noninfectious RV-like particles (VLPs) containing the three structural proteins of RV in BHK (baby hamster kidney cells) cell line is outlined in Qui et al., Journal of virology 1994 (4086-4091).
However, producing RV-antigens in eucarotic cell systems is labour intensive and time consuming, while the yield is comparatively low. Establishing of in vitro diagnostic methods of anti-rubella virus antibody detection requires procedures of producing recombinant RV antigens from procaryotic organisms like E. coli in a defined, soluble, functional, and reproducible quality with clear advantages compared to the established RLPs.
Although the know-how in the field of protein-design and engineering is strongly increasing, the rubella antigens E1, E2 and C are expressed with very low abundance in E. coli host cells and, moreover, they are poorly soluble.
The immuno-dominant rubella antigen, i.e. the antigen of choice for the immunological detection of antibodies from human sera, is the envelope-protein E1. The overall topology of the E1 protein comprises a large extracellular ectodomain (1-452) including an—helical region (438-452) with a transmembrane domain (453-468) followed by a short C-terminal anchor-segment (until 481).
One reason for the insolubility of E1 of RV could be due to the conformation of the ectodomain which is supposed to be stabilized by ten disulfide bridges. The rubella E1 peptide is organized into an amino-terminal (N-terminal) region and a carboxy-terminal (C-terminal) region with an overall content of 24 cysteine residues.
In general, the N-terminal end refers to the extremity of a protein or polypeptide terminated by an amino acid with a free amine group (NH2), while the C-terminal end refers to the extremity of a protein or polypeptide terminated by an amino acid with a free carboxyl group (COOH).
Gros et al., (1997) disclose that the N-terminal region of the rubella E1 protein contains eight disulfides constructed from the cysteine residues C(8) to C(287), while the C-terminal region contains two disulfides generated from the cysteine residues C(249) to C(401). The residues C(456), C(466), and C(468) are located in the predicted transmembrane segment, and residue C(470) is predicted to be located in the interior of the virus. These data indicate that the residues C(456) to C(470) are not involved in disulfide formation.
The wealth of cysteine residues with the concomitant likelihood of false bridging events (intramolecular and intermolecular SH-shuffling is very probable to occur) suggests that the conformational and oxidative refolding of E1 is an extremely complex process which is difficult to control in vitro. Therefore, there is an urgent need for a simple and reliable protocol which facilitates both purification and high-yield refolding of the rubella antigens into an immunoreactive (i. e. antigenic) conformation. Native-like folded recombinant E1 envelope variants are a mandatory requirement to achieve a reliable immunoassay for antibodies against RV.
The rubella E1 ectodomain (1-452) contains 20 cysteine-residues. Their correct bridging determines the three-dimensional structure and is crucial for the exposition of the correct, native-like conformational epitopes. The oxidative refolding of cysteine-rich proteins, that is the formation of the correct intramolecular and/or intermolecular disulfide bridges, is a complex and experimentally demanding process. For the insertion of disulfide bridges in vivo into de-novo-synthesized or translocated proteins an arsenal of folding helpers (chaperones, oxido reductases, prolyl isomerases) are available in cell compartments (endoplasmic reticulum in eucaryotic, periplasm in procaryotic cells). Since the redox potential of the bacterial cytosol is strongly reducing (−270 mV), the cysteines of heterologous target proteins naturally occur as thiol moieties. When the expression is very high, the target proteins are deposited in the host cell as insoluble aggregates (the so-called inclusion bodies, IB).
Usually, the partially refolded and unfolded protein-fraction is solubilized under reducing conditions (e.g. 5 mM TCEP) in chaotropic solutions (7.0 M GdmCl, 8.0 M urea) resulting in unfolded protein chains, which have to be renatured (i.e. refolded into the native or a native-like conformation). As a general rule the yield of native-like folded protein is strongly decreased by incorrect disulfide bridges.
Disregarding conformational aspects, even limited numbers of cysteines (and the assumption of strictly intramolecular bridging events) yield huge numbers of erroneous bridging events ending up in unproductive side reactions like aggregation processes.
Different from in-vitro-refolding in a test tube, a huge arsenal of folding helpers in an optimized redox ambience as folding-assistance are available for the de novo folding in vivo. Nevertheless, the soluble expression of functional cysteine-rich proteins in procaryotic host cells like E. coli is still very complex. The aim to enhance the overproduction rate of immunoreactive antigens of RV and to increase the solubility of the refolded proteins has been achieved only to a limited extent.
In Newcombe et al., Clin Diagn Virol. 1994 (3)149-63, nine gluthatione-S-transferase (GST) E1 fusion proteins were used to express rubella E1 antigen-fragments in E. coli in a soluble form. Only after a substantial truncation of the E1 sequence a successful soluble expression was possible for the cysteine-free region 243-286 (44 amino acid residues). EP 299,673 discloses a peptide from amino acid residues 207-353 which retains rubella Ig specific binding characteristics.
Furthermore, Starkey et al., J. Clin. Microbiol. (1995), 270-274) disclose that only a very narrow area of 44 to 75 amino acid residues of a GST-E 1 fusion protein were soluble. GST fusion proteins containing the entire E1 coding sequence and larger subfragments were expressed in forms which could not be purified and were therefore presumed to be expressed as insoluble inclusion bodies.
In order to identify immunoreactive determinants within RV antigens, the E1 and E2 epitopes have been mapped extensively in the past years by using synthetic peptides (Mitchell et al., Virus Research 29(1993), 33-54). In addition, distinct independent epitopes have been located within RV E1 protein including domains that are important for viral infectivity and hemagglutination (Waxham and Wolinsky, Virology (1985) 153-65, Green and Dorsett, J. Virol. (1986) 57, 893-898, Ho-Terry et al., Arch Virol. 1985; 84 (3-4):207-15. The recombinant protein A-El-fusions described by Teffy et al, Arch Virol. (1988), 98, 189-97, characterized solely linear epitopes. The closer localization of epitopes of RV E1 glycoprotein is described by Terry et al., (1988), Wolinsky et al., J Virol. (1991), 3986-94, and Chaye et al., J Clin Immunol. 1993 Mar; 13(2) 93-100. Furthermore, Gieβauf et al., J. Immun. Meth. 287 (2004), 1-11, evaluate an ELISA by using several coated peptides of E1 to improve the determination of immunity against rubella. Only one of these peptides, the BCH-178 peptide, seemed to be successful for screening neutralizing antibodies as an additional method to confirm low positive or borderline HAI titres or RV-IgG values. There is no further proof that the small BCH-178 peptide (amino acid residues 213-239) contains most of the antigenic epitopes of the native E1 molecule (SEQ ID NO: 4). However, no reference has been made until now for the localization of the main reactivity of the immundominant rubella E1 envelope protein.
It was the aim of the present invention to provide a soluble rubella E1 variant from procariotic cell systems, which reconciles high solubility and high immunological reactivity in a serological assay.